JOURNAL OF BACTERIOLOGY, Sept. 1991, p. 5516-5522 0021-9193/91/175516-07$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 173, No. 17

Purification and Characterization of Escherichia coli Heat-Stable Enterotoxin II YOSHIO FUJII,1 MITSUO HAYASHI,2 SHUNJI HITOTSUBASHI,l YASUNORI FUKE,l HIROYASU YAMANAKA,' AND KEINOSUKE OKAMOTO'* Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho,

Tokushima

770,1 and Research Center of Toyo Jozo, 632-1, Mifuku, Ohito, Shizuoka 410-23,2 Japan Received 27 February 1991/Accepted 19 June 1991

Escherichia coli heat-stable enterotoxin II (STIU) was purified to homogeneity by successive column chromatographies from the culture supernatant of a strain harboring the plasmid encoding the STII gene. The purified STII evoked a secretory response in the suckling mouse assay and ligated rat intestinal loop assay in the presence of protease inhibitor, but the response was not observed in the absence of the inhibitor. Analyses of the peptide by the Edman degradation method and fast atom bombardment mass spectrometry revealed that purified STII is composed of 48 amino acid residues and that its amino acid sequence was identical to the 48 carboxy-terminal amino acids of STU predicted from the DNA sequence (C. H. Lee, S. L. Moseley, H. W. Moon, S. C. Whipp, C. L. Gyles, and M. So, Infect. Immun. 42:264-268, 1983). STII has four cysteine residues which form two intramolecular disulfide bonds. Two disulfide bonds were determined to be formed between Cys-10-Cys-48 and Cys-21-Cys-36 by analyzing tryptic hydrolysates of STII.

(17), contains the STII gene derived from pCG86 and also carries the chloramphenicol resistance gene. Culture supernatants of E. coli strains harboring pCHL7 and pWM501 are known to be positive in the STII assay (11, 17). SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli (9), with a gradient polyacrylamide gel (gradient of 4 to 22.5% polyacrylamide). The samples were heated for 10 min at 100°C in the presence of 2% 2-mercaptoethanol and 2% SDS and applied to the gels. Electrophoresis was performed at a constant current of 20 mA for 3 h. The gels were stained with Coomassie brilliant blue and then destained as described in the laboratory manual for the LKB 2117 Multiphor II electrophoresis system (LKB Produkter AB, Bromma, Sweden). Methanol treatment of culture supernatant. E. coli HB101 harboring appropriate plasmids was cultured in 2 ml of Luria broth containing ampicillin (50 ,ug/ml) for pCHL7 and pBR322 or chloramphenicol (50 ,ug/ml) for pWM501 at 37°C overnight with shaking. The supernatant was separated from the cells by centrifugation. A 10-fold excess of analyticalgrade methanol was added to the supernatant. The suspension was allowed to stand at 4°C overnight. The resulting precipitates were collected by centrifugation at 30,000 x g for 1 h. After being washed with methanol, the precipitates were dried under vacuum and dissolved in 40 ,ul of distilled water. The materials obtained were designated methanol precipitates. Crude sample preparation. The gene product of the STII gene was purified from the culture supernatant of E. coli HB101(pCHL7). The cells were grown in Luria broth containing ampicillin (50 jig/ml) at 37°C overnight with shaking. The supernatant was separated from the cells by centrifugation. Ammonium sulfate was added to the supernatant (40 g/100 ml). STII was precipitated from the supernatant by the addition of ammonium sulfate. The resulting precipitate was recovered by centrifugation and then dissolved in distilled water. The protein concentrations of the samples were adjusted to 14 mg/ml. Methanol was poured slowly into the samples to a final concentration of 60%. The precipitates

Enterotoxigenic Escherichia coli strains produce two kinds of heat-stable enterotoxin (STs), which cause intestinal secretion and diarrhea (1, 6, 23). One of the proteins is termed STI (also referred to as STa), and the other is STII (also referred to as STb). STI is methanol soluble and active in the suckling mouse assay and the porcine ligated ileum model (2, 23). STI has been well characterized chemically, and the nucleotide sequence of the STI gene has been determined (20-22). The amino acid residues involved in the expression of STI activity have been determined (4, 5, 15, 19), and a mode of action has been proposed (18). On the other hand, STII is methanol insoluble and active only in the weaned-pig ligated ileum model (2, 7, 24). The gene encoding STII has been cloned and its nucleotide sequence has been determined (10, 11, 17), but little is known about the biological and physiocochemical properties of STII because the method for the purification of STII has not been established and purified STII has not been obtained. Recently, Kupersztoch et al. reported that mature STII is a 48-amino-acid extracellular polypeptide that corresponds to the inferred carboxy-terminal amino acid sequence (8), but the purification procedure for and the enterotoxicity of the 48-amino-acid peptide have not been shown. Therefore, it is not clear that the extracellular 48-amino-acid peptide is active STII. In this article, we describe the procedures for purification of STII and its biological and physicochemical properties. MATERIALS AND METHODS Bacterial strains and plasmids. E. coli HB101 was used as the host strain in all experiments and was cultured in Luria broth (13). Plasmids pCHL7 and pWM501, which carry the E. coli STII gene, were kindly provided by H. W. Moon and W. K. Maas, respectively. Plasmid pCHL7, approximately 5.7 kb in size (11), was constructed by insertion of the 1.3-kb Hindlll-Hindll fragment of P307 at the Hindlll site of pBR322. Plasmid pWM501, approximately 6.4 kb in size *

Corresponding author. 5516

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which formed were composed mainly of ammonium sulfates. As STII was not precipitated by this methanol treatment, the precipitates were removed by centrifugation at 15,000 x g for 20 min. The supernatants obtained were concentrated twofold by rotary evaporation under vacuum at 40°C. The solutions prepared in this manner were designated crude

samples. Column chromatography. Crude sample was applied to a column (2 by 40 cm) of DEAE-Sephadex A-25 (Pharmacia LKB Biotechnology, Uppsala, Sweden) equilibrated with 10 mM Tris-HCI buffer (pH 7.5). Materials were washed with 100 ml of the same buffer, and elution was performed with a 500-ml linear gradient of 0 to 1 M NaCl in the same buffer. Fractions containing STII were collected and then applied to a column (2 by 40 cm) of SP-Sephadex C-50 (Pharmacia LKB Biotechnology) equilibrated with 10 mM Tris-HCI buffer (pH 7.5). After the adsorbent was washed, materials were eluted with 450 ml of a linear gradient of 0 to 1 M NaCl in the same buffer. The active fractions obtained from SP-Sephadex column chromatography were further purified by reversed-phase column chromatography on Pep by the fast protein liquid chromatography system (Pharmacia LKB Biotechnology). The elution was performed with a gradient of 0 to 70% acetonitrile in 0.05% trifluoroacetic acid (TFA). The preparations obtained were designated purified STII. Before application of the purified protein to SDS-PAGE, the samples (20 jig) were treated with 2% SDS in the presence or absence of 2% 2-mercaptoethanol at 100°C for 3 min. Sample preparation for enterotoxic activity assays. The enterotoxic activity of the purified STII was assayed in a suckling mouse assay and a rat intestinal ligated loop assay. Reports have indicated that STII does not show enterotoxic activity in rats and suckling mice because STII administered into the intestinal lumen of these animals is degraded by intestinal proteases (24). However, a recent report showed that the intestinal response to STII is observed in these animals when protease inhibitors are present to block the intestinal protease activity (25). Aprotinin (Bayer, Levenkusen, Germany) was used as a protease inhibitor to examine the enterotoxicity of the purified STII in this experiment. The purified peptide was dissolved in saline containing 100 U of aprotinin per ml, and the mixture was administered to the experimental animals as described below. The amounts of purified STII used for enterotoxic activity are indicated in Fig. 6 and Table 1. In the rat intestinal ligated loop assay, samples containing 12.5, 25, 100, or 200 p,g of purified STII were injected into each of the rat intestinal loops; in the suckling mouse assay, 10 and 40 ,ug of purified STII was administered. Protein content was determined by the method of Lowry et al. (12) with bovine serum albumin as a standard. Enterotoxic activity assays. The methods used for rat intestinal loop assays were basically those of Whipp (24, 25). Sprague-Dawley rats weighing 250 to 350 g were used. Rats were deprived of food (but not of water) for 48 h before the experiment. After sodium pentobarbital anesthesia, a series of ligated intestinal segments (loops), about 5 cm long and separated by a 1- to 2-cm interloop, were created. The most proximal loop was placed about 5 cm distal to the ligament of Treitz. Each loop was injected with 0.5 ml of purified STII. The sample activity was determined postmortem (3 h after injection) and expressed as the weight of loop (in grams) per length of loop (in centimeters). The suckling mouse assay was performed as described previously (14, 16). Briefly, 0.1-ml samples were adminis-

CHARACTERIZATION OF E. COLI STII

5517

tered by gastric tube into the stomachs of 2- to 3-day-old suckling mice with 0.001% Evans blue dye as a marker. The mice were killed 2.5 h later, and ratios of intestine weight to body weight were determined. An intestine/body weight ratio of 0.083 was considered a positive response. Amino acid composition of purified STII. Samples dissolved in water were hydrolyzed in 6 M HCl at 1100C for 24 h in vacuum-sealed tubes, and the resulting hydrolysates were analyzed by a type 835 amino acid analyzer (Hitachi Ltd., Tokyo, Japan). Tryptophan was not determined because it is destroyed by the hydrolysis procedure. Cysteine was not analyzed quantitatively by this method. No additional attempts were made to determine the amounts of tryptophan and cysteine. Peptide sequence determination of purified STII. Sequence analyses of peptides were carried out by the Edman degradation method with a protein gas phase sequencer connected to a phenylthiohydantoin analyzer (PSQ-1 system; Shimadzu Ltd., Kyoto, Japan). The apparatus was operated according to the manufacturer's instructions. The degradation was performed in 49 steps. Since the peptide was not alkylated before being sequenced, at positions where unidentified signals were obtained, cysteine may be the actual amino acid present. FAB mass spectrometry of purified STII. Fast atom bombardment (FAB) mass spectra were recorded with a JMSSX102 mass spectrometer equipped with a JMA-DA6000 data system (JEOL, Tokyo, Japan). The samples were dissolved in 10% acetic acid mixed with 5% thioglycerol and 5% glycerol and applied to a stainless steel sample holder. The conditions used in these experiments were as follows: xenon atom beam source, 3-keV accelerating potential and 20 mA emission current, and 4-kV accelerating potential ion source. Enzymatic digestion and fragment separation. Purified STII (500 ,ug) was incubated with 25 ,ug of trypsin type III (Sigma, St. Louis, Mo.) at an STII/enzyme ratio of 20:1 (wt/wt). After a 15-h incubation at 36°C (pH 7.5), the reaction was stopped by boiling the mixture for 10 min. The reaction mixture was separated by high-pressure liquid chromatography (HPLC) on a C18 reversed-phase column (Cosmosil 10 C18-300; Nakarai, Kyoto, Japan). The separation revealed that purified STII was cleaved into nine fragments by trypsin. Each of the fragments eluted from the reversed-phase column was collected and subjected to the same analyses used for intact purified STII. Statistical analyses. Statistical analyses of data were performed with Student's t test. RESULTS Detection of the STII gene product. It is known that STII is secreted into the culture supernatant and that the toxin in the supernatant is methanol insoluble (2, 8). In order to detect a peptide unique to an E. coli strain producing STII, the methanol precipitates prepared as described in Materials and Methods were analyzed on SDS-PAGE. E. coli HB101 carrying pBR322 was used as a control. A picture of a gel after staining with Coomassie brilliant blue is shown in Fig. 1. As shown by the arrow in Fig. 1, a 5,100-molecular-weight band was detected in the lanes in which the samples from both E. coli HB101(pCHL7) and E. coli HB101(pWM501) were run, but the corresponding band was not observed in the lane of the sample obtained from E. coli HB101 (pBR322). These observations indicate that the 5,100-molecular-weight peptide is STII.

5518

FUJII ET AL.

J. BACTERIOL. 0.2 .0

i

io!. ::.,

sQ

0.1

i.5 z'U

.0 :.

(kDa) 16.9

,--

1-

i

It,

0

-

14.4 6.2 2.5 -

40

20

0

60

80

100

120

Fraction number

1 2 3 4 FIG. 1. SDS-PAGE analysis of methanol precipitates prepared from culture supernatants of E. coli harboring either pBR322 (lane 2), pWM501 (lane 3), or pCHL7 (lane 4). Preparation of the methanol precipitates and analysis of the proteins are described in the text. Low-molecular-weight markers (Pharmacia LKB Biotechnology) were used as molecular weight size standards (lane 1). The arrow indicates the position of putative STII.

Purification of putative STII. The 5,100-molecular-weight peptide was purified from the culture supernatant of E. coli HB1O1(pCHL7). SDS-PAGE was used to detect the putative STII. The crude sample was prepared as described in Materials and Methods. The majority of the putative STII in the culture supernatants was recovered in the crude sample. A result of chromatography of the crude sample on DEAE-Sephadex A-25 is shown in Fig. 2, where putative STII emerged in the area shown by the horizontal bar. The fractions containing the peptides were then subjected to cation-exchange chromatography on SP-Sephadex C-50. The peptide was recovered in fractions eluted with 0.4 M NaCl (Fig. 3). The final purification step involved reversed-phase column chromatography on Pep (Pharmacia LKB Biotechnology). Putative STII was eluted as a sharp single peak with about 38% acetonitrile (Fig. 4). The peptide obtained by this chromatography was used as purified STII. The purified STII gave a single band on SDS-PAGE (Fig. 5). The mobility of the purified peptide on SDS-PAGE was changed by treatment with 2-mercaptoethanol, as shown in

FIG. 3. SP-Sephadex C-50 column chromatography. The peptide fractions obtained from the DEAE-Sephadex A-25 column were applied to an SP-Sephadex C-50 column. Materials were washed with about 150 ml of 10 mM Tris-HCl buffer (pH 7.5) and then eluted with 450 ml of a linear gradient of 0 to 1 M NaCl in the same buffer. Fractions (5 ml) were collected and assayed for A280. The heavy horizontal bar indicates the fractions pooled for subsequent purification.

Fig. 5, indicating that the peptide possesses intramolecular disulfide bonds. Enterotoxic activity. The enterotoxic activity of the purified peptide was examined in the presence of aprotinin in both a rat intestinal loop assay and a suckling mouse assay. The results are shown in Fig. 6 and Table 1. More than 100 ,ug of the purified peptide caused obvious fluid accumulation in the rat intestinal loop assay. In the suckling mouse assay, the purified peptide (40 ,ug of protein) caused a positive response. However, the same amount of peptide did not show enterotoxic activity in the absence of aprotinin (data not shown). These results demonstrate that the purified peptide is active STII and that in vivo responses to STII are influenced by the peptidases in the intestinal lumen. Amino acid analysis of purified STII. The amino acid composition of purified STII was determined as described in Materials and Methods, and the results are shown in Table 2. As the number of cysteine and tryptophan residues was not accurately measured by the method used in this experiment, values for these amino acid residues are not shown. The composition of purified STII was identical to that of the 48 carboxy-terminal amino acid residues of STII predicted from the DNA sequence (11, 17). Amino acid sequence and molecular weight. The amino acid -

E

-60

c

2.(

0

-1

00 cl

0

-

-1

1.01

0

70

-1

50

I

-

.*

- 40

ev

,:

-E

30

c

4

r~~~~~~~~~~~~~~

0.5

.0

20

z I I

-1 IA

Ia

U () 2()

4() 60Ii)In(X) (

F;raction number

FIG. 2. DEAE-Sephadex A-25 column chromatography. Crude sample was applied to a DEAE-Sephadex A-25 column equilibrated with 10 mM Tris-HCl buffer (pH 7.5). Fractions (5 ml) were collected and assayed for A280. The heavy horizontal bar indicates the fractions pooled for subsequent purification.

5

10

15

20

25

30

35

40

45

50

Time (min)

FIG. 4. Reversed-phase HPLC of the peptide obtained from an SP-Sephadex C-50 column. Chromatographic conditions: solvent A, 0.05% TFA; solvent B, 70% acetonitrile in 0.05% TFA; flow rate, 0.5 ml/min. The gradient program was: 0 to 5 min, 0% acetonitrile; 5 to 8 min, 0 to 20% acetonitrile; 8 to 12 min, 20% acetonitrile; 12 to 50 min, 20 to 70% acetonitrile.

VOL. 173,

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~;.-

2

CHARACTERIZATION OF E. COLI STII

TABLE 1. Purified peptide-induced fluid secretion in the suckling mouse assay Peptide used' (p.g)

Mean secretory response + SE (intestine/body weight)

No. of mice

0 (control) 10 40

0.052 ± 0.002 0.065 ± 0.004b 0.084 ± 0.003c

8 11 11

" The indicated amounts of peptide dissolved in 0.1 ml of aprotonin solution were administered to suckling mice as described in the text. b p < 0.05 compared with control. ' P < 0.0002 compared with control.

16.9

14A 62 25

1

FIG. 5. SDS-PAGE of purified peptide. Before application, samples (20 ,ug) were treated with 2% SDS in the presence (lane 1) or absence (lane 2) of 2% 2-mercaptoethanol at 100°C for 3 min. Low-molecular-weight markers were as described in the legend to Fig. 1.

sequence of purified STII was analyzed by the Edman method. The sequence determined was identical to the 48-carboxy-terminal amino acid sequence of mature STII predicted from the DNA sequence (Fig. 7). Purified STII was analyzed by FAB mass spectrometry (Fig. 8). The mass value (5104.28) is almost identical to the theoretical value (5104.87) calculated from the protonated molecular formula of C216H351067N66S5, in which four cysteine residues form two disulfide bonds. These results indicate that the molecular weight of STII is 5103.86 (the value obtained by subtracting the proton molecular weight from the theoretical value) and that two disulfide bonds are formed intramolecularly. Mode of disulfide bond formation. Mature STII contains four cysteine residues, one each at positions 10, 21, 36, and 48. These cysteine residues are thought to be linked intramolecularly by two disulfide bonds. To determine the location of disulfide bonds, purified STII was digested with trypsin,

and the resulting hydrolysates were separated by reversedphase HPLC on a Cosmosil 10 C18 column. Elution, performed with a linear gradient of 0.1% TFA and 45% acetonitrile in 0.1% TFA, produced seven peaks, designated 1, 2, 3, 4, 5, 6, and 7. The elution profile is shown in Fig. 9A. The area under peak 4 was larger than that under the others, indicating that peak 4 was composed of more than two tryptic hydrolysates. Peak 4 was further separated by HPLC on the same column with a linear gradient of 5 to 25% acetonitrile in 0.1% TFA. Three peaks appeared and were designated 4-1, 4-2, and 4-3 (Fig. 9B). The nine fragments of STII were subjected to gas phase amino acid sequencing. Phenylalanine residues are observed as the carboxy-terminal amino acid residues in fragments 4-2 and 5. This indicates that the trypsin used in this experiment is contaminated with chymotrypsin, but the analyses were not affected by the contamination. Sequencing of the nine fragments revealed that four fragments (3, 4-1, 4-2, and 5) contained two peptide chains (Table 3). Since these chains invariably include cysteine residues, it is likely that these two chains are linked by TABLE 2. Amino acid composition of purified E. coli STII Amino acid

Ala Arg Asx Cys Glx

Gly His

Ile Leu Lys Met Phe Pro

Ser Thr Trp Tyr Val (O

Si)

1(x)

lS()

2)(

tIoxin (.g)

FIG. 6. Fluid accumulation induced by purified peptide in the rat intestinal loop assay. The indicated amount of purified peptide was dissolved in aprotinin solution (protease inhibitor) and injected into rat intestinal loops. The amount of accumulated fluid in the loops

examined 3 h later as described in the text. Data are means standard error for three to five separate experiments.

was

5519

No. of residuesa

Determined by hydrolysis

Nearest integer

5.56 1.87 2.85

6 2 3 (4) 5 6

c

5.00 5.60 0.95 1.82 2.00 5.53 0.79 1.79 NDd 2.84 1.93 0.91 1.83

1

2 2 6 1 2 0 3 2 (0) 1 2

No. predicted from DNA sequenceb

6 2 3 4 5 6 1 2 2 6 1 2 0 3 2 0 1 2

" Compositions are expressed as the number of residues per molecule of E. coli STII (molecular weight, 5,100). Numbers in parentheses are taken from the last column because the amounts of corresponding amino acids were not determined by the method used. b The amino acid composition of the 48 carboxy-terminal amino acids of the predicted E. coli STII from DNA sequence (11, 17). '-, not determined. d ND, not detectable.

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FUJII ET AL.

J. BACTERIOL.

1

Met Lys Lys Asn lie Ala Phe Leu LIu Ala Ser Met Phe Val Phe Ser Ile Ala Thr Asn Ala Tyr Ala Ser Thr 10

Gin Ser Asn Lys Lys Asp Lcu (Cys) Glu His Tyr Arg

Gin lie Ala Lys Glu Ser (Cys) Lys Lys Gly Phe leu Gly 30

4

-

A

B4-1

U I

40

Val Arg Asp Gly Thr Ala Gly Ala (Cys) Phe Gly Ala Gln 48

lie Met Val Ala Ala Lys Gly (Cys)

f

FIG. 7. STII amino acid sequence as inferred from the DNA sequence (11, 17) and determined (underlined) amino acid sequences of purified STII determined by the Edman degradation method. Amino acids at positions where unidentified signals were obtained by this method were inferred to be cysteines, as described in the text. These cysteine residues are shown in parentheses.

5

7I

.1

qu

4-2

/4-3

disulfide bonds. The regions of the intact STII from which these chains were derived could be inferred by contrasting the amino acid sequence of each fragment with that of intact STII. The inferred regions and the probable amino acid sequence of each chain are shown on the right side of Table 3. The results proposed the mode of disulfide bond formation, that is, disulfide bridges were supposed to be formed between Cys-10 and Cys-48 and between Cys-21 and Cys-36. To confirm the location of the disulfide bonds, three fragments, 4-1, 4-2, and 5, were subjected to analysis by FAB mass spectrometry and amino acid composition analysis. Fragment 3 was not analyzed because the domain covered by fragment 3 was almost the same as that covered by fragment 4-1. The results obtained are shown in Tables 4 and 5. The theoretical molecular weight of each fragment was estimated from the presumed sequence shown in Table 3. Identity between the mlz value obtained by FAB mass spectrometry (MH value) and the theoretical molecular weight value was observed (Table 4). The differences between the two values come from the fact that free peptides give a mass peak of protonated molecular ions in the FAB mass spectrometry. The amino acid compositions of these fragments were found to be identical to that of the presumed sequence except for the tyrosine in fragment 4-1 (Table 5). The tyrosine in fragment 4-1 was probably destroyed during hydrolysis preceding analysis. These results demonstrate that disulfide bonds are formed between Cys-10-Cys-48 and

Cys-21-Cys-36. DISCUSSION We purified STII to homogeneity from the culture supernatant of E. coli cells transformed by pCHL7, which contains STII gene, by ammonium sulfate fractionation and ~~~~~~~~51 289

R R

5t5

dS1B

A60-

d40 X:"I i u~~~uk

5

S0e58

519

5150

5200 M,z

FIG. 8. FAB mass spectra of purified STII. The threshold for analyzing peaks was set at 20.

3

0

10

20

30

in) Time (mian)}

40

S0

0

10

Time (fi)

20

FIG. 9. Reversed-phase HPLC separation of tryptic hydrolysates of STII. (A) Hydrolysates were applied to a Cosmosil 10 C18 column, and the peptides were separated by a linear gradient of acetonitrile containing 0.1% TFA from 0 to 45% in 45 min. (B) The peak 4 fraction shown in panel A was further separated on the same column under different conditions as described in the text. The sample was applied to a column equilibrated with 5% acetonitrile containing 0.1% TFA and eluted by a linear gradient of acetonitrile containing 0.1% TFA from S to 25% in 40 min. A part of the chromatogram is shown.

successive column chromatographies. The identity of purified STII was established by amino acid and biological activity analyses. Although STII has been reported to produce water secretion only in the piglet intestinal loop assay (2, 24), it was recently reported that STII evokes an intestinal fluid secretory response in the suckling mouse assay and rat intestinal loop assay when endogenous protease activities are blocked with protease inhibitors (25). The peptide purified according to the method presented above evoked a secretory response in the suckling mouse assay and rat intestinal loop assay when mixed with protease inhibitor (aprotinin) (Table 1 and Fig. 6). These results demonstrate that the purified peptide is biologically active STII. The enterotoxic activity of the purified STII could not be observed in the absence of aprotinin. This indicates that the purified STITs are degraded by the proteases in the intestinal lumen. The biological activity of purified STII was not consistent in that there was dispersion among the accumulated volumes in intestines in our experiments (Fig. 6). Furthermore, more STII than STI was required to evoke an intestinal response (21, 22). These results may be due to the intraluminal factors that preclude a secretory response of STII, such as proteases not inhibited by aprotinin. Whipp reported that the intestinal response to STII was dose related in rats and that the experimental errors became small when the intestinal lumen was rinsed with saline (25). It is possible that the reactions of the purified STII would become consistent and the amount of STII required to evoke the response would become lower if the intestinal lumen were rinsed. Subsequently, we analyzed the amino acid composition and sequence of purified STII. The analyses showed that purified STII is composed of 48 amino acid residues and that

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CHARACTERIZATION OF E. COLI STII

5521

TABLE 3. Amino acid sequences of tryptic hydrolysates Fragment

1

2

Gly Lys

3

Amino acid residue at positiona 3 4 6 7 5

(Cys) Asp Leu

(Cys)

Glu

His

Tyr

Glu

His

Tyr

Arg

Asp Leu (Cys)

4-1

Gly

4-2

5

a

The amino acid

Arg

Thr

Ala

Glu

(Cys)

Lys

Lys

Gly

Ser

Asp Gly

Thr

Ala

Glu Ser

(Cys)

Lys

of each fragment

was

Gly

Ala

(Cys)

Phe

47

Gly- Cys

48

Asp30 Gly31 Thr32 Ala33 Gly34 Ala35 Cys36 Ph37 1

Ala

(Cys)

Phe

Asp! Gly-l Thr32 Ala-3 Gly4-19 Ala-3 Cys-6 Phe 20

G s21 Glu-SerCys-Lys

22

determined by Edman degradation.

TABLE 4. Molecular weight of tryptic hydrolysates

Theoretical value' m/z[MH]b

12 13 14 7 8 9 ~10 I11 Lys-Asp-Leu9-Cys-Glu-His-Tyr-Arg 1

19 20 G GIuSerCys 21- Lys 22-yYS23

its amino acid sequence coincides fully with the 48 carboxyterminal amino acids of STII inferred from the DNA sequence (Fig. 7). The molecular weight of 5103.86 estimated from the sequence has now been confirmed by FAB mass spectrometry (Fig. 8). From the STII DNA sequence, it has been predicted that STII is synthesized as a 71-amino-acid residue precursor (11, 17). The 23 amino-terminal amino acid residues inferred from the DNA sequence were absent in purified STII (Fig. 7). These observations confirm the recent report by Kupersztoch et al. (8) that mature STII is composed of the 48 carboxy-terminal amino acid residues. As they discussed, the 23-amino-acid residue amino-terminal peptide is thought to be cleaved by a signal peptidase. It is important to define the reactive site(s) on the protein in order to determine the mechanism of action of the protein. To define the site(s), the tertiary structure must be determined. An initial determinant of the structure of a protein is intramolecular disulfide bonds. The results of SDS-PAGE and FAB mass spectrometry indicated that there are two intramolecular disulfide bonds in STII. We tried to determine the locations of the disulfide bonds by analyses of tryptic hydrolysates of STII. The results demonstrate that the bonds are formed between cysteines at positions 10 and 48 and positions 21 and 36 (Table 3). Determination of the

Mol wt of fragment:

Determination

Probable amino acid sequence and position in the sequence of mature STII

Asp-Leu- Cys- Glu- His -Tyr- Arg Gly - Cys

(Cys)

Asp Gly

sequence

8

4-1

4-2

5

1,110 1,111

1,331 1,332

1,203 1,204

a Calculated from the presumed amino acid sequence shown in Table 3. b Mass values were determined by FAB mass spectrometry.

mode of disulfide bond formation should contribute to further studies of STII. Recently, Dubreuil et al. reported the purification of STII from the culture supernatant of a porcine E. coli strain (3). The molecular weight of their purified STII was estimated to be 5,000 by urea-SDS gel electrophoresis. The value is almost identical to our determined value, showing that both toxins are the same. The STII produced by the porcine E. coli strain existed as a high-molecular-weight aggregate which was dissociated by urea (3). We have not observed such aggregates. It is possible that the concentrations of salt

TABLE 5. Amino acid compositions of tryptic hydrolysates Amino acid

Asx Thr Ser Glx Gly Ala Val Cys Met Ile Leu Tyr Phe Lys His Arg

No. of residues (no. predicted)a in fragment: 4-1 4-2 5

1.1 (1) 0 (0) 0 (0) 1.2 (1) 1.2 (1) 0 (0) 0 (0) NDb (2) 0 (0) 0 (0) 1.0 (1) 0.01 (1) 0 (0) 0 (0) 0.9 (1) 0.9 (1)

1.0 (1) 0.9 (1) 0.9 (1)

1.1 (1) 2.0 (2) 2.0 (2) 0 (0) ND (2) 0 (0) 0 (0) 0 (0) 0 (0) 0.9 (1) 1.9 (2) 0 (0) 0 (0)

1.0 (1) 0.9 (1) 0.9 (1) 1.1 (1) 2.0 (2) 2.0 (2) 0 (0) ND (2) 0 (0) 0 (0) 0 (0) 0 (0) 0.9 (1) 1.0 (1) 0 (0) 0 (0)

a Numbers in parentheses are from the presumed amino acid sequence shown in Table 3. b ND, not determined.

5522

FUJII ET AL.

and STII in the samples affect the formation of aggregates. Further studies are necessary to clarify the process of forming the aggregates. ACKNOWLEDGMENTS We thank S. Lehnhardt for critical reading of the manuscript. This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture, Japan.

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